1. Field of the Invention
The present invention relates to the promotion of the growth of granulocytes from human bone marrow utilizing nerve growth factor.
2. The Prior Art
Recently a nerve growth factor was derived from mouse saliva and mouse submandibular glands, which is characterized by its high degree of stability in dilute aqueous solutions and a molecular weight of 116,000 (Orenstein et al (1978) Proc. Natl. Acad. Sci., U.S.A. 75, 5497-5000, U.S. Pat. Nos. 4,185,095 and 4,407,744). The factor has several uses, i.e., plasminogen activator (U.S. Pat. No. 4,185,095), neurite outgrowth stimulator (U.S. Pat. No. 4,185,095), wound healing (U.S. Pat. No. 4,281,184; Li et al (1980) Proc. Natl. Acad. Sci., U.S.A. 77, 4379-4381), substitutes enzymically for the first component of complement (Boyle et al (1982) Proc. Natl. Acad. Sci., U.S.A. 79, 2519-2522), leukocyte chemotaxis mediator (Lawman et al (1984) J. Immunol. Methods, 69, 197-205; Boyle et al (1984) J. Immunol., in press; and Gee et al (1983) Proc. Natl. Acad. Sci., U.S.A. 80, 7215-7218). An antibody to the mouse nerve growth factor has also been elicited (U.S. Pat. No. 4,230,691).
This mouse derived nerve growth factor (NGF) has a molecular weight of 116,000, contains 1 gram-atom of tightly bound Zn(II) per mole (Young et al (1980) Biochemistry 19, 5316-5321; Pattison et al (1975) Biochemistry 14, 2733-2739), and consists of three different subunits, termed .alpha., .beta., and .gamma. (Varon et al (1967) Biochemistry 7, 1926-1303). The .beta.-subunit is responsible for promoting neurite outgrowth from sympathetic and embryonic sensory ganglia (Levi-Montancini et al (1968) Physiol. Rev. 48, 534-569; Bocchini et al (1969) Proc. Natl. Acad. Sci. U.S.A. 64, 787-794). The .gamma.-subunit is a member of the serine class of proteolytic enzymes (Orenstein et al, supra; Thomas et al (1981) J. Biol. Chem. 256, 9156-9166), and displays a highly restricted substrate specificity towards certain lysyl and argininyl peptide bonds (Orenstein et al, supra; Greene et al (1968) Proc. Natl. Acad. Sci. U.S.A. 60, 1383-1388). The .alpha.-subunit has no known function. Recent studies have shown that the subunit structural formula for NGF is .alpha..sub.2 .beta..gamma. (Young et al, supra). The molecular weight of all of the subunits lie in the range 26,000-28,000.
In an area of research totally unrelated to nerve growth factor, several agents have been discovered which stimulate growth of granulocyte colonies from primitive bone marrow stem cells. Known granulocyte colony-stimulating factors (G-CSF) include those disclosed by Metcalf et al (1983) J. Cell. Physiol. 116, 198-206; Nicola et al (1983) J. Biol. Chem. 258, 9017-9023 and Burgess et al (1977) J. Biol. Chem. 252, 1998-2003.
The mouse lung-derived granulocyte stimulating factor (Nicola et al, supra) is a glycoprotein of molecular weight 25,000 and contains internal disulfide bonds. Mouse lung G-CSF exhibits half-maximal activity upon normal mouse marrow cells at 3.times.10.sup.-12 M. Whether it possesses a protease activity is not known. At low concentrations, this G-CSF stimulates only granulocyte colony-forming cells. At higher concentrations it promotes the growth of mixed macrophage plus granulocyte colonies, as well as small numbers of macrophage colonies.
The known G-CSF factors are obtainable only in extremely small amounts thereby greatly limiting the avenues of research necessary to establish practical therapeutic and diagnostic applications for these agents. For example, research on the normal granulocyte maturation process as well as in pathologic states such as leukemia wherein the maturation process is altered would be greatly facilitated by the ready availability of large amounts of a G-CSF.
It is an object of the present invention to provide a novel composition and method for stimulating the growth of granulocytes from primitive bone marrow stem cells which are based upon factors possessing heretofore undiscovered G-CSF activities which are readily obtainable in large amounts.